We provide evidence that the putative tumor suppressor function of miR-449a is, in part, dependent on Rb in prostate cancer cells. DU-145 cells devoid of wild-type Rb are resistant to cell cycle arrest and senescence induced by miR-449a. Only upon restoration of Rb did miR-449a regain its growth inhibitory effects in the DU-145 sublines. Based on our data, a simple model can be created linking miR-449a to Rb activation and growth arrest in prostate cancer cells (Figure ). We show that miR-449a can regulate Rb phosphorylation by directly targeting Cyclin D1 and HDAC1. Modulation of Rb activity through HDAC1 knockdown is likely facilitated through downstream targets such as the activation of p27 expression. Interestingly, p27 directly binds cyclins (e.g. Cyclin D1) and inhibits CDK4/6 complex activity [28
]. By this mechanism, miR-449a has a dual approach for regulating Cyclin D1 activity and Rb phosphorylation.
A model linking miR-449a to Rb activation and growth arrest in prostate cancer cells
It has also been reported that miR-449a directly targets and represses the expression of CDK6 and CDC25A; two more key factors involved in promoting Rb phosphorylation [29
]. Further evidence has indicated that miR-449a is also transcriptionally regulated by E2F transcription factors [29
]. E2F proteins preferentially interact with hypophosphorylated Rb, which sequesters their activity. Upon Rb phosphorylation, E2F proteins are released to activate downstream gene expression. Because E2F family members function as downstream mediators of Rb, miR-449a functions in an auto-regulatory loop to control Rb activity by targeting multiple upstream regulatory factors (i.e. Cyclin D1, HDAC1, CDK6, and CDC25B). Collectively, these reports, in combination with our data, define miR-449a as an integral miRNA component of the Rb pathway.
Phenotypically, miR-449a is a multifaceted miRNA in that it induces apoptosis in association to cell cycle arrest [16
]. However, we reveal that miR-449a promotes apoptosis in prostate cancer cells regardless of Rb status; both PC-3 and DU-145 cells are susceptible to the apoptotic effects of miR-449a. Previous research has indicated that the specific knockdown of HDAC1 contributes to the apoptotic effects of miR-449a [16
]. Because HDACs regulate numerous downstream factors including apoptotic genes (i.e. Bcl-XL, etc.), miR-449a may promote apoptosis in an Rb-independent manner through depletion of HDAC1 [32
]. In addition, miR-449a may also directly target and suppress the expression of antiapoptotic genes. For instance, in silico
analysis utilizing the miRanda algorithm reveals a putative target site in the 3'UTR of the BCL2 transcript [25
]. We also reveal that wild-type Rb enhanced the apoptotic response in DU-145 sublines to suggest that miR-449a may facilitate apoptosis through Rb-dependent mechanisms, as well. In support, Rb activation is known to trigger apoptosis by sequestering E2F protein activity and repressing the expression of downstream antiapoptotic gene BIRC5/survivin [34
Precise control of Rb activity is absolutely essential for maintaining regulated cellular growth. In nearly all cancer types Rb is inactivated to promote oncogenesis. We propose that the loss of miR-449a expression can promote Rb inactivation and prostate cancer progression. In vivo
analysis has shown that miR-449a is depleted in human prostate tumor tissue relative to patient-matched controls [16
]. Furthermore, miR-449a is located in a chromosomal region previously identified as a susceptibility locus in a variety of malignancies including prostate cancer [35
]. The mechanism by which miR-449a is depleted in prostate cancer may result from genomic deletion or epigenetic silencing. Co-treatment of histone methylation and HDAC inhibitors has been shown to re-activate miR-449a expression in breast cancer cells [29
]. Regardless, loss of miR-449a would disrupt its auto-regulatory control over Rb and promote unregulated growth, which may, in part, contribute to transformation during prostate cancer tumorigenesis. Our data supports the tumor suppressor-like function of miR-449a by highlighting its relationship with Rb and describing its inhibitory effects on cell growth. Although miR-449a-mediated cell cycle arrest is largely Rb-dependent, re-activation or replacement of miR-449a may have therapeutic benefit in prostate cancer that retains functional Rb status.